Principal Investigator/Program Director (Last, First, Middle): Michel, Thomas PROJECT 1: "Insulin and Receptor-Modulated Pathways of eNOS Regulation." Thomas Michel, Project Leader Abstract: This project will explore the mechanisms by which insulin and related receptor pathways regulate the endothelial isoform of nitric oxide synthase (eNOS) and associated signaling proteins in vascular endothelial cells. This project interacts at multiple levels with each of the other projects in this Program, not only in view of the central role of endothelium-derived NO and insulin signaling in normal vascular homeostasis, but also on account of their importance in arterial dysfunction, oxidative stress, and diabetic vasculopathy. In response to the activation of diverse cell surface receptors[unreadable] including the insulin receptor[unreadable] NO is synthesized by the eNOS. The fundamental hypothesis of this research program is that derangements in eNOS and in related endothelial pathways contribute to the abnormalities in NO-dependent signaling that have been identified in diabetes. We have discovered that eNOS undergoes reversible nitrosation in endothelial cells; Aim 1 will determine the roles of nitrosation in the regulation of eNOS. We plan to identify the cysteine residue(s) in eNOS that undergo reversible nitrosation; characterize receptor-mediated pathways involved in eNOS nitrosation and denitrosation; elucidate the effects of eNOS nitrosation on enzyme activity; explore the consequences of eNOS phosphorylation and subcellular targeting on enzyme nitrosation; and determine the relationships between diabetes, insulin signaling and eNOS nitrosation. Aim 2 of this project will explore the role of the scaffolding protein caveolin-1 in insulin-mediated signal transduction in endothelial cells. Following siRNA-mediated knockdown of caveolin in cultured endothelial cells, insulin-dependent phosphorylation of the pluripotent kinase glycogen synthase kinase 3-p (GSK- 3-P) is markedly enhanced. By contrast, the abrogation of caveolin-1 expression by siRNA markedly attenuates basal activity and receptor-dependent regulation of Rho in endothelial cells. These studies will define the mechanisms whereby caveolin inhibits the activation of Pho GTPases and GSK- 3p. Studies in Aim 3 will explore the mechanisms whereby insulin potentiates sphingosine 1-phophate (S1P) signaling in vascular endothelial cells. We will identify the pathways whereby insulin induces synthesis of the S1 P! receptor, and will extend these studies to analyze arterial preparations from diabetic animals. Our studies of the cellular and molecular mechanisms whereby insulin regulates eNOS and related signaling pathways may lead to the identification of new points for pharmacological intervention in diabetic arteriopathy.